The polymerase chain reaction (PCR) method is a technique to amplify a nucleic acid by cloning the base sequence thereof, and there are an endpoint PCR and a real-time PCR. Hitherto, the endpoint PCR has been often used, but by this method, it is possible to detect or quantify the amplified nucleic acid only after the PCR reaction is completed, and thus there is a problem that a separate result analysis step such as electrophoresis and an apparatus for that are required after the experiment, a long time of 2 hours or longer is required, and it is not possible to detect or quantify the amplified nucleic acid in real time. For example, the first nucleic acid is required to be quantified from the PCR product obtained after the reaction is completed, but it is difficult to accurately quantify the first nucleic acid due to various factors which affect the analysis even if fluorescent staining or the like is employed. The accurate amount of a nucleic acid is measured at the exponential phase, but it is difficult to analyze the accurate amount by the endpoint PCR since a real-time measurement is impossible by it.
In contrast, the real-time PCR has an advantage that the first target nucleic acid can be accurately quantified since the amount of the amplified nucleic acid is measured for every cycle and particularly the reaction in the exponential phase that is the section in which the amplification takes place can be confirmed in real time through a monitor. In particular, the multiplex real-time nucleic acid amplifying method (multiplex real-time PCR) is widely used in the disease diagnostic field since it is possible to confirm various biomarkers in a single chamber by one time of experiment and to quantitatively analyze them in real time.
However, it is difficult to conduct an accurate measurement by a method that is the most commonly used method for multiplex real-time nucleic acid amplification and uses the color of the probe and the melting point of the primer since the interference between the targets increases as the number thereof to be measured increases. Hence, it is difficult to use the multiplex real-time nucleic acid amplifying method in the fields that require an accurate diagnosis of diseases through simultaneous and rapid analysis of many different kinds of nucleic acids such as point-of-care technology (POCT).